Methods for testing the concentration of an analyte in a body fluid

ABSTRACT

Devices and methods for utilizing dry chemistry dye indicator systems for body fluid analysis such as glucose level in whole blood are provided by incorporating a porous membrane with a skin side which enables separation of whole blood and visually reading the indicator without removing the red blood cell portion of the blood from the membrane. The devices also enable visual reading of the indicator by use of a cellulose fiber glass composite matrix which provides separation of whole blood in a lateral flow of the blood through the matrix from the input area to a test area of the matrix. Another aspect of the device provides a determination of hematocrit level in whole blood by measuring flow rate through a restricted passageway in the device.

FIELD OF THE INVENTION

The present invention relates to a test device and method for thecalorimetric determination of a chemical or biochemical component(analyte) in an aqueous body fluid, such as whole blood. In particularthe present invention relates to a dry reagent test strip from which ananalyte presence and/or concentration is determined by visualinterpretation or through the use of an instrument. A common use of suchtest strips is for determination of glucose level in blood by diabetics.

BACKGROUND OF THE INVENTION

Numerous devices have been developed to test for presence and quantityof analytes in aqueous samples, such as whole blood or urine. The patentand technical literature of the last thirty years is replete withinventions which utilize a reagent strip containing a dry chemistryreagent system, that is, a system in which the wet chemistries areimbibed into an absorbent or bibulous medium, dried, and laterreconstituted by fluid from the test sample. The reagent strips containan indicator which changes color, depending on the presence orconcentration of a particular analyte in a biological fluid applied tothe strip. These strips may be read visually by reference to a colorstandard or calorimetrically by instrument calibrated or programmed todetect a certain color. Although some of these strips use reductionchemistries, more commonly they involve an oxidizable dye or dye couple.Some of the strips include an enzyme, such as glucose oxidase, which iscapable of oxidizing glucose to gluconic acid and hydrogen peroxide.They also contain an oxidizable dye and a substance having peroxidativeactivity, which is capable of selectively catalyzing oxidation of theoxidizable dye in the presence of hydrogen peroxide. (See, for example,U.S. Pat. No. 5,306,623, to Kiser et al.) Examples of these devices, inaddition to those used to test blood glucose, include tests forcholesterol, triglycerides, calcium or albumin in whole blood, and forprotein, ketones, albumin or glucose in urine.

Dry chemistry reagent strips incorporating enzyme-based compositions areused daily by millions of diabetics to determine blood glucoseconcentrations. The NIH sponsored study, the Diabetes Complications andControl Trial, demonstrated conclusively that careful control of bloodglucose levels can significantly reduce the incidence of seriouscomplications of diabetes such as vision loss and kidney malfunction.Most diabetics must test themselves periodically in order to makeappropriate adjustments to their diet or medication. It is thusespecially important for diabetics to have rapid, inexpensive, andaccurate reagent strips for glucose determination. The embodiment of drychemistry reagent systems in test strips enable simple yet effectiveanalytical protocols.

The technologies embodied in the products which have been developed todate have certain limitations from the perspective of the end userand/or the manufacturer. There is, therefore, a need to overcome some ofthe limitations of currently available colormetric testing systems.

U.S. Pat. No. 3,092,465, issued to Adams et al., U.S. Pat. No.3,298,789, issued to Mast and U.S. Pat. No. 3,630,957, issued to Rey etal., all describe a basic reagent system which became a standard forcolorimetric determination of glucose in biological samples. Thesepatents describe the formation of a film layer or semi-permeable coatingover the bibulous matrix to hold back the larger particulates, such asred blood cells, and allow fluid to permeate into the bibulous matrix.This approach requires the removal of red blood cells by washing orwiping to enable visual inspection or instrument reading of theindication of the dye color formed in the matrix.

Stone, U.S. Pat. No. 3,607,093, discloses a membrane for testing bloodwhere the membrane has a skin permeable to solutions but impermeable tosolids such as red blood cells and to macromolecules such as proteins.This membrane is disclosed as being used by applying a blood sample thenwiping away the red blood cells from the skin in order to reach the testindication through the skin.

U.S. Pat. No. 3,552,928, issued to Fetter discloses the use of certainwater soluble salts and amino acids in reagent formulations asseparation agents to provide blood separation. With solids such as redblood cells substantially removed from the biological fluid, there isless background color at the test site to obscure a change in colorationproduced by a testing reagent.

Phillips et al., U.S. Pat. No. 4,935,346 discloses a system wherein awhole blood sample is applied to the device and indicator developmentoccurs in the presence of the colored components of the sample.Measurements of the color change in indicator are made at two distinctwavelengths to eliminate the interferences from the presence of coloredblood components.

Kiser et al., in U.S. Pat. Nos. 5,306,623 and 5,418,142, disclose avisual meter device which incorporates various coatings on a matrixmaterial to filter red blood cells from fluids. Similar devices forvisual indication are disclosed by Hochstrasser in U.S. Pat. Nos.3,964,871 and 4,059,407.

Terminello et al., U.S. Pat. No. 4,774,192, disclose a system in whichthe matrix is formed of an asymmetric material used to filter the redblood cells in the sample. The asymmetric material has a densitygradient from one side to the other to progressively separate red bloodcells from the fluids.

Daffern et al., U.S. Pat. No. 4,994,238, disclose a test device thatcomprises an asymmetric reagent layer that has progressively finerfiltration with increased distance from one surface toward the othersurface.

Castino et al., U.S. Pat. No. 5,456,835 disclose the use of filtersformed of ligand modified polymeric film such as polypropylene fibersand polyethersulfone fibers.

Vogel et. al., U.S. Pat. No. 4,477,575, disclose the use of glass fibermaterial to achieve blood separation through the thickness of thematerial. Blood is applied to one side of the glass fiber, andrelatively clear fluid migrates out of the opposite side. This fluid isdelivered to an additional layer where the detection of analytes canoccur.

Macho et al., U.S. Pat. No. 5,451,350, disclose the use of absorbentchannels to distribute sample fluid in multi-zone test devices. Charltonet al., U.S. Pat. No. 5,208,163, also disclose the use of capillarychannels to distribute blood to various chambers in the device.

The disclosures of the above patents are incorporated herein byreference.

The prior art devices and methods of the above references providevarying degrees of effectiveness of blood analysis at varying degrees ofcomplexity and cost.

It is an object of the present invention to provide improved devices andmethods to improve the performance and minimize the cost and complexitycompared to the prior art devices.

It is a further object of the present invention to provide a fullydisposable, discrete reading system for detecting analyte presence orconcentration.

It is another object of this invention to provide a dry reagentchemistry system capable of analyzing whole blood for one or moreanalytes without prior separation of the red blood cells from the serum.

It is another object of this invention to provide a means for performingmicrotitration for the analysis of whole blood in a system which enablesthe ready visual determination of analyte presence or concentration.

It is yet another object of this invention to provide a blood separationsystem which can be used with a dry chemistry reagent to analyze wholeblood for one or more analytes.

It is still a further object of this invention to provide a drychemistry reagent and test strip which can be used in an electronicmeter to analyze whole blood for one or more analytes.

The above objects as well as others are achieved by the devices, methodsand systems of this invention as disclosed herein.

SUMMARY OF THE INVENTION

In one aspect this invention provides a method of testing blood for thepresence or concentration of an analyte by using a porous matrixcomprising a skin side and a test side, wherein the skin side comprisesa porous skin capable of blocking the passage of red blood cells and ofallowing passage of blood fluids containing an analyte to the test sideof the matrix, and wherein the test side of the matrix is isotropic foruniform distribution therein of fluid received from the skin side andcomprises an indicator capable of indicating the presence orconcentration of the analyte. The method comprises applying a bloodsample to the skin side of the matrix, allowing the fluid to passthrough the skin into the isotropic matrix, then reading or measuring onthe test side of the matrix the indication provided by the indicator ofthe presence or concentration of the analyte without removal of the redblood cells from the skin side of the matrix. The skin side isoptionally treated with compounds which assist in blocking the passageof red blood cells and allowing passage of substantially clear fluid.Such compounds, or separating agents, can help facilitate the wicking ofthe clear fluid into the test side of the matrix. However, it ispreferred that the skin side of the matrix is inherently hydrophilicwhich facilitates the passage of fluid through the skin to the test sideof the matrix while blocking passage of the red blood cells. Thisseparation of the blood on the skin side and reading or measuring theresultant indication on the test side of the matrix makes thedetermination of the presence and/or concentration of analyte simplerdue to the relative absence of red blood cells at the test site ofsystem and due to the absence of the necessity of removing the red bloodcells before taking the desired reading or measurement.

In another aspect this invention provides a device for testing blood forthe presence or concentration of an analyte comprising a holdercomprising an opening for receiving a blood sample; and a porous matrixcomprising a skin side and a test side wherein the skin is capable ofblocking the passage of red blood cells and of allowing the passage ofblood fluids containing an analyte to the test side of the matrix andwherein the test side of the matrix is isotropic for uniformdistribution of fluid received from the skin side. The test side of thematrix comprises an indicator for indicating the presence orconcentration of an analyte in the fluid. The matrix is attached to theholder so that the skin side is oriented toward the opening in theholder for receiving the blood sample such that when a blood sample isapplied in said opening the blood contacts the skin side of the matrixallowing the blood fluids to pass to the test side of the matrix and redblood cells to be retained on the skin side of the matrix. The devicecan optionally have a support member applied to the test side of thematrix, where the support member has a visual opening through which theindicator is read or measured.

Alternatively, the support member can be a solid layer, and the holdercan have a second opening through which the indicator can be read ormeasured after the fluid passes through the skin and into the matrixextending under the second opening in the holder. In this alternative,the skin side and the test side can be on the same side of the matrixmember, but the skin providing the blockage of red blood cells is in adifferent area from the test area of the matrix. In such an alternative,an adequate seal is provided to prevent whole blood from flowing fromthe skin area to the test area but only allow blood fluids to passthrough the skin to the test side or area of the matrix.

In another aspect this invention provides a method of making a devicefor testing blood for the presence of an analyte comprising providing aholder comprising an opening for receiving a blood sample and laminatingto the holder a porous matrix comprising a skin side and a test sidewherein the skin is capable of blocking passage of red blood cells andof allowing passage of blood fluids containing an analyte to the testside of the matrix and wherein the test side of the matrix is isotropicfor uniform distribution of fluid received from the skin side. In thisembodiment, the skin side of the matrix is in contact with the holderand the opening in the holder communicates with the skin on the skinside of the matrix.

In another aspect this invention provides a device for testingconcentration of an analyte in a fluid sample comprising a first membercomprising an opening having a predetermined volumetric size and aporous matrix member positioned within said opening in the first memberfor receiving an amount of fluid to fill the volumetric opening. Thematrix member comprises an indicator capable of indicating the presenceof the analyte, and the matrix member comprises a skin side and a testside wherein the skin side is capable of blocking the passage of solidspresent in the fluid and of allowing passage of fluid containing ananalyte to the test side of the matrix positioned in the volumetricopening. It is preferred that the skin side of the matrix member is amaterial which is inherently hydrophilic and facilitates the passage ofthe fluid through the skin side to the test side of the member. Thedevice can optionally have a support member with a visual opening atleast in part aligned with the opening in the first member whereby thefluid sample can be applied to one opening, the skin can block passageof solids but allow passage of fluid to the test side of the matrix andthe analyte can be detected in the test side of the matrix through theother opening. Sequentially or simultaneously the predeterminedvolumetric size of the opening in the first member provides for aquantitative measurement of the concentration of the analyte in thefluid by enabling titration of a known amount of indicator reagent and agiven volumetric quantity of fluid containing the analyte and the colorindicator provides a qualitative indication. This invention furthercomprises methods of using these devices to quantitively measure ananalyte in a fluid.

In another aspect this invention provides a method of making a devicefor testing concentration of an analyte in a fluid comprising providinga first member being substantially noncompressible and having an openingtherein of a predetermined volumetric size and providing a porous matrixmember which is fluid permeable and is compressible compared to thefirst member. The method comprises pressing the matrix member againstthe first member so that a portion of the matrix member protrudes withinsaid opening and a portion of the matrix member is compressed againstthe surface of the first member adjacent to said opening. Optionally, asupport member with an opening aligned with the opening in the firstmember can be laminated to the first member to position the compressedportion of the matrix between the first member and the support member.Also, optionally the compressed portion of the matrix member can beremoved leaving the portion of the matrix member within the opening. Thematrix member used in this method of making such devices optionally canhave a skin side wherein such a matrix member is positioned in thedevices as described above wherein the skin side protrudes into saidopening or the skin side faces the support member.

In another aspect this invention provides a device for the testing forthe presence or concentration of an analyte in a fluid sample comprisinga first member comprising an opening for receiving a fluid sample, aporous matrix member positioned in communication with and extendinglaterally from said opening in the first member, where the matrix membercomprises an initial area, which is in communication with the opening inthe first member, and a test area, which is a given distance laterallyfrom the initial area. The matrix member contains pores which arecapable of blocking in the lateral distance between the initial area andthe test area the passage of solids in the fluid sample and capable ofallowing passage of fluid the lateral distance from the initial area tothe test area of the matrix. The test area of the matrix comprises anindicator capable of indicating the presence or concentration of theanalyte. This device can optionally comprise a support member comprisingan opening therein on which support member the first member and thematrix member are mounted so that the matrix member is positionedbetween the first member and the support member and so that the openingin the support member is offset from the opening in the first member andis positioned over at least a portion of the test area of the matrix.This device is capable of receiving a fluid through one opening at theinitial area of the matrix, allowing the fluid to pass laterally throughthe matrix from the initial area of the matrix to the test area of thematrix while the pores of the matrix provide blocking of the passage ofsolids. The other opening at the test area of the matrix is a visualopening which allows detection of the indication of the indicator.Alternatively, the second opening at the test area of the matrix membercan be in the first member at the given lateral distance from theopening at the initial area of the matrix. The optional support membermay be solid with no openings. In this alternative device, the fluidsample is received in the first opening at the initial area and theindicator read at the second opening at the test area where bothopenings are on the same side of the device.

In the above embodiments utilizing lateral flow of the fluid, ananisotropic or asymmetric porous matrix can be used. For example, insuch a matrix separation of solid components can occur based ondecreasing or changing pore size in the matrix. However, in suchembodiments an isotropic porous matrix may be employed where uniformsized pores block the passage of solids. In either case, the solids suchas red blood cells, introduced at the initial area of the matrix can beheld back from the test area of the matrix. If the solids are notadequately blocked and are allowed close to the test area, the solidsmay cast a shadow or cause color difference in the test area of thematrix. In such cases compensation may need to be made in the reading ofthe indicator.

In another aspect this invention provides a device for testing for thepresence or concentration of an analyte in a fluid sample comprising amember having a first opening for receiving a fluid sample and a secondopening for receiving fluid from the first opening wherein the firstopening and the second opening are connected by a restricted flowpassageway or delivery channel communicating with the first opening andsecond opening thus enabling the fluid sample to flow from the firstopening to the second opening through the restricted flow passageway.This device further comprises a detector for detecting and measuring therate of initial flow of the fluid from the first opening through therestricted flow passageway towards the second opening. This aspect ofthe invention also provides a method of using such device wherein therate of initial flow of fluid through the restricted passageway ismeasured and correlated to the concentration of a particularconcentration of solids (e.g., hematocrit level) in the fluid sample. Ithas been found that the rate of initial flow of fluid through therestricted flow passageway can be directly correlated to theconcentration of an analyte and the fluid. Optionally, in this aspect ofthe invention the second opening may contain a porous matrix positionedin the second opening comprising an indicator for indicating thepresence or concentration of an analyte in the fluid sample entering thematrix. Also, optionally, the porous matrix positioned in the secondopening may comprise a skin side and a test side as described above inconnection with other embodiments of this invention. In this aspect ofthe invention the measurement of the rate of initial flow of the fluidthrough the restricted flow passageway can also be correlated to theindication provided by the indicator in the matrix in the second openingthus providing more complete information with respect to the hematocritlevel of the fluid. Also optionally a matrix material may be present inthe restricted flow passageway or delivery channel, and the initial flowrate therethrough can be correlated to an hematocrit level of the bloodas described above.

The above embodiments of the devices of the invention with theappropriate dry chemistry system in the matrix member can be used intest strips which may be read visually or measured in an electronicmeter. Electronically read devices or strips are provided withappropriate calibration data and test initiation sequences which can beincorporated on the strips in the form of bar codes, digital punches,magnetic signals or the like. These codes or signals on the test stripsprovide appropriate data to the meter and eliminate the need for inputsfrom the user. These aspects simplify the test protocol and reduce thepotential for user generated error.

The above sets forth the generic aspects of the various devices andmethods of the present invention. These devices and methods are morefully described in the drawings and the descriptions below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective exploded view of a matrix member and a memberhaving and opening for receiving a fluid.

FIG. 2 is a perspective view of the device of FIG. 1 assembled.

FIG. 3A shows an electronic meter receiving a device of FIG. 2 and adrop of fluid entering the device of FIG. 2.

FIG. 3B shows an example of machine readable coding on the device ofFIG. 2

FIG. 4 is a perspective exploded view of a matrix member positionedbetween a first member and a support member, where the device comprisesa plurality of test sites for one analyte or multiple analyte tests.

FIG. 5 is a perspective view of the device of FIG. 4 assembled.

FIG. 6 is a perspective exploded view of a device having a plurality oftest sites for one or more analytes and a delivery channel fordelivering fluid from a central sample introduction point to a pluralityof test sites.

FIG. 7 is a perspective view of the device of FIG. 6 assembled.

FIG. 8A is a perspective exploded view of a matrix member partiallycompressed and partially protruding into an opening in a noncompressiblemember.

FIG. 8B is a perspective exploded view of a matrix member partiallycompressed between a first member and a support member and partiallyprotruding into an opening in the first member.

FIG. 9 is a perspective view of the device of FIG. 8B assembled.

FIG. 10A is a perspective exploded view of the device of FIG. 8B havinga plurality of test microtitration sites for one analyte or multipleanalyte tests.

FIG. 10B is a perspective view of the device of FIG. 10A assembled.

FIG. 11 is a perspective view of a partially compressed matrix withrounded protrusions for extension into openings.

FIG. 12 is a perspective exploded view of a device of FIG. 10A having aplurality of microtitration test sites for one or more analytes and adelivery channel for delivering fluid from a central sample introductionpoint to a plurality of test sites.

FIG. 13 is a perspective exploded view of the device of FIG. 12assembled.

FIG. 14 is a perspective exploded view of the device of FIG. 12 whereinthe test sites are arranged to provide gravity-aided flow of the fluidsample to the test sites. FIG. 15 is a schematic diagram of thecapillaries and test sites of the device in FIG. 14.

FIG. 16 is a schematic diagram of the openings in the device in FIG. 14.

FIG. 17 is a perspective view of a noncompressible member having anopening which can be pressed together with a compressible matrix memberto partially compress the compressible matrix member and cause theprotrusion of a portion of a matrix member into the opening of thenoncompressible member.

FIG. 18 is a perspective view of the partially compressed matrix memberafter removal from the noncompressible member.

FIG. 19 is a perspective view of a shaped matrix insert formed byremoval of the compressed portion of the article of FIG. 18.

FIG. 20 is a perspective exploded view of a device having a plurality ofindividual matrix inserts of FIG. 19 placed in openings in a firstmember having a delivery channel for delivering fluid to each of theopenings from a central sample introduction point.

FIG. 21 is a perspective exploded view of a matrix member positionedbetween a first member having an opening and a second member having anopening offset from the opening in the first member.

FIG. 22 is a perspective view of the device of FIG. 21 assembled.

FIG. 23 is a perspective exploded view of a matrix member partiallycompressed and partially protruding into an opening in a noncompressiblemember positioned between a first member having an opening and a secondmember having an opening offset from the opening in the first member.

FIG. 24 is a perspective view of the device of FIG. 23 assembled.

FIG. 25 shows a device according to FIG. 21 having a plurality of testsites for one analyte or multiple analyte tests.

FIG. 26 is a perspective view of the device of FIG. 25 assembled.

FIG. 27 is a perspective exploded view of the device of FIG. 23 having aplurality of test sites for one analyte of multiple analyte tests.

FIG. 28 is a perspective exploded view of the device of FIG. 25 having adelivery channel for delivering fluid from a central sample introductionpoint to a plurality of test sites.

FIG. 29 is a perspective view of the device of FIG. 28 assembled.

FIG. 30 is a perspective exploded view of the device of FIG. 27 having adelivery channel for delivering fluid from a central sample introductionpoint to a plurality of test sites.

FIG. 31 is a perspective exploded view having a flow rate determinativedelivery channel from a sample introduction opening to an openingcontaining an optional matrix member.

FIG. 32 is a perspective view of the device of FIG. 31 assembled andoptical detectors for measuring the flow rate of fluid moving throughthe delivery channel.

FIG. 33 is a perspective exploded of the device of FIG. 31 wherein thedelivery channel contains a matrix member.

FIG. 34 is a perspective exploded view of a device similar to FIG. 20wherein the delivery channel is formed on the backside of the membercontaining the sample introduction opening.

FIG. 35 is a perspective view of the device of FIG. 34 assembled.

FIG. 36 is a bottom view or view of the backside of the membercontaining the sample introduction opening and showing the deliverychannel in the device of FIG. 34.

FIG. 37 is a perspective view of a test strip of FIG. 14 and showinguser instruction on the back of the strip at the blood applicationpoint.

FIG. 38 is a front view of the test strip illustrated in FIG. 37 showinguser indicia for indicator readings.

FIG. 39 is a perspective exploded view of the device of FIG. 4containing an individual or discrete matrix member for each fluidreceiving opening and test site.

DETAILED DESCRIPTION OF THE INVENTION

The devices of the present invention are simpler to use and are easierand less costly to manufacture than most devices previously available.This is especially important for diabetics who rely on blood glucosetesting multiple times per day to keep their disease under control. Formany diabetics, the costs associated with blood glucose monitoring aresignificant, especially elderly diabetics on fixed incomes. Devices ofvarious configurations and various uses based on the embodiments of theinvention disclosed herein can be delivered to the diabetic patient, ina more cost effective manner. The ease of use and portability of thesedevices, coupled with more attractive pricing, will facilitate increasedpatient compliance with recommended testing routines and will result inimproved overall health of diabetic patients.

In one or more aspects this invention uses an intrinsically hydrophilicmembrane and takes advantage of and enhances the blood separationcapabilities of such a membrane. This invention includes separating thewhole blood and employs a microtitration system so that the separatedclear fluid can be analyzed independently of the red blood cells. Thissegregation or isolation from the red cells of the clear fluid beinganalyzed is necessary to eliminate interferences from the highly coloredcells and provide a more consistent liquid sample for the titration ofthe analyte by eliminating the majority of the blood solids from thetest area. The red blood cells can mask the color indication of theindicator reagent making it difficult or impossible to read. If wholeblood is absorbed into the test areas, volumetric differences due tovarying solids content in the blood affect the titration sample sizewhich can result in an inaccurate measurement of the analyte. Byseparating whole blood according to this invention into red blood cellsand substantially clear fluid, an accurate analysis can be obtained onboth a qualitative and quantitative basis. As used herein, reference isprimarily made to blood. However, other fluids such as urine, saliva andthe like can be analyzed utilizing the various embodiments of thepresent invention.

The invention uses membranes from two categories. The first categoryincludes microporous membranes which separates the blood solids fromblood fluids. The most preferred microporous membranes arepolyethersulfone polymeric membrane which is formed with a skin sidewhich acts as a red blood cell barrier and a matrix side which hasuniform pore size for containing indicator reagents. The second categoryincludes cellulose glass fiber composites or polymer based membrane ormatrix products which facilitate lateral wicking of fluid and provideseparation of blood solids from blood fluids. Vertical separation occursperpendicular to the application side, through the depth of thematerial. Lateral separation occurs within the membrane parallel to thesurface of the application side. In either category, this inventionprovides devices which avoid the necessity for meter reading. Due to theseparation of red blood cells, these devices provide reliable visualreading of the indicator by the user. The improved separation and visualreading is in part provided by the devices of this invention where theblood solids and red blood cells are maintained in a floating state onthe skin side or in some cases in the lateral matrix, which assists inkeeping the color from the solids and cells from contaminating the testareas where visual reading of the indicator is desired.

The first membrane type can be treated with separation agents and testreagents. In a preferred embodiment, the membrane is inherentlyhydrophilic, has a smooth skin side and a rough matrix side which is anisotropic porous matrix. The whole blood is applied to the skin side andthe combination of skin characteristics, hydrophilic matrix andseparation agents hold the red blood cells on the surface of the skinside while clear fluid and analytes flow into the matrix. The key isthat the whole blood must be delivered from the skin side to achieveproper separation. This mechanism creates a titration area in the matrixarea free of red blood cells and containing a consistent volume ofrelatively clear fluid. The hematocrit effect normally found in drychemistry tests is minimized as long as adequate clear fluid is provided(by the highest hematocrit blood specified) to rehydrate the indicatorreagents while the red blood cells are blocked by the skin from enteringthe matrix. A reservoir is preferably provided for the sample so thatupon separation of whole blood and relatively clear fluid, a largeenough volume of fluid is provided to the solids in the matrix so theyare fully hydrated, even with a high hematocrit blood where some excessof fluid remains on the skin surface of the membrane or within thereservoir.

The membrane of the first type are preferably a polyethersulfone polymerwhich is cast to inherently have a microporous skin on one side and aporous matrix on the other side, such as the Gelman membrane. However,one may also employ a matrix layer having uniform porosity but nobarrier skin on either side by laminating to one side of such a matrix amicroporous barrier film to form the required barrier skin on one sideof the matrix.

Membranes of the second type are also preferably treated with separationand test reagents. The whole blood is applied to an initial area of thematrix, and the matrix wicks the fluid laterally to a test area of thematrix. As it wicks out, the separation reagents enhance the separationof the whole blood into red blood cells and relatively clear fluid. Thematrix is preferably a naturally hydrophilic material. As the bloodseparates, clear fluid moves from the initial zone into the test zoneand reacts with the indicator reagents to indicate the presence andconcentration of analyte. The test zones must be positioned such thatclear fluid migrates into the zones without red blood cells. In otherwords, for the highest hematocrit blood specified, there must be enoughclear fluid to migrate to the test area to activate and react with theindicator reagent system. This invention minimizes the hematocrit effectobserved in some test devices. Providing a uniform and adequate samplevolume assures a uniform hydraulic head at each test site. The quantityof relatively clear fluid is such that, although the reservoir containsboth red blood cells and clear liquid, the test volume supplied is theproper volume of sample for testing.

The invention provides different mechanisms for using the dry chemistryreagent systems with and without microtitration volume control. The drychemistry components and microtitration principles are described below,independent of the embodiments which follow.

The microtitration concept employed in some aspects of this inventioncan be explained as a method of controlling the sample volume and thereagent amount to give a consistent titration and therefore consistentand reliable results. The first step is to create a test zone which isbounded. The traditional wet chemistry analysis uses a fixed(premeasured) volume of sample and titrates a quantity of test reagentagainst that sample. In a dry format the quantity of the test reagenthas to be impregnated into the matrix in a ratio proportional to thevoid volume of the matrix. This can be accomplished many different ways.The sample volume (SV) is the void volume of the matrix (VVM) minus thesolids volume remaining in the matrix from the test reagent followingwet application and drying or test reagent volume (TRV). The ratioSV/TRV must be constant to provide an accurate titration.

To achieve microtitration the material void volume and the reagentapplication must be controlled. The device of this invention creates afixed control geometry which does not permit cross talk between testareas and the sample delivery channel. The microporous membrane has atendency to wick laterally, which the device in this aspect of theinvention prevents. The whole blood is delivered so that it enters thetest area matrix from the skin side of the microporous material. Thesample may be introduced in any orientation to the laterally wickingmaterials which may alternatively be employed. The glass fiber materialbecomes quite fragile when fully wetted. Therefore, it is practical toonly impregnate reagents in the test zones. This can be accomplished byusing a syringe or needle to discretely apply the reagents in the testarea. The most effective way to do this is to preassemble the device andcoat the reagents while the cellulose and glass fiber is supported bythe front panel of the test strip device. The other materials can beimpregnated into the matrix either locally or by general application butin a controlled fashion.

In this invention, the preferred method for controlling the test areageometry is to emboss the membrane into the gasket or molded part,deforming a portion of the membrane into openings in the gasket ormolded part and leaving the test areas uncompressed and compressing aportion of the membrane. The compressed areas are fastened to the gasketwith adhesive such as 3-M grade 415 acrylic pressure sensitive adhesive,creating test areas which are completely bounded on the sides whichprevents any flow between. The only means of sample entry into eachopening is through the top, i.e., the skin side (e.g., see FIG. 10A).The membrane is embossed into the gasket by bringing both piecestogether between two platens of a hydraulic press which pushes a portionof the membrane into the gasket openings and deforms the materialoutside of the openings by compressing it so that the thickness isreduced by 80 to 95% in the compressed area. (See FIG. 8B)

The material which is embossed can be die cut and the compressed arearemoved (in a process similar to creating a label on a printing press,see FIGS. 17-19) to eliminate any chance for cross talk between testzones. In this embodiment the test zones are held to the device only bya small ring of adhesive; the majority of the embossed or compressedmaterial having been removed. The adhesive seals to the gasket memberinto which the die cut matrix inserts are inserted thereby preventingany leakage of fluid between test zones.

A second method can be utilized to create the microtitration zones. Thismethod, shown in FIGS. 8A and 8 B, is also similar in concept tocreating labels. An individual microtitration zone is attached to aviewing window or is captivated in a gasket. Adhesive is applied to anonporous element in the area where the test zone is desired. A viewingwindow is punched in the nonporous member leaving an annular ring ofadhesive. A sheet of membrane is applied to the part and laminated tothe nonporous member at the adhesive rings. A die then cuts the membranearound the viewing hole and slightly greater in diameter than theadhesive ring. The unattached membrane is peeled away, leaving the testzones attached to the nonporous member at the viewing windows. (See FIG.20.)

The sample can enter the microtitration zones via openings in a gasketlayer which are fed by a capillary passageway formed in a separatelayer. Alternately, the gasket and capillary may be molded as a singlepiece of material. A wetting agent may be applied to the bottom of thecapillary channel to facilitate blood flow without the presence of anabsorbent material in which the sample may run. High molecular weightpolymeric oils work well as wetting agents. A preferred material isdimethylsiloxane ethylene oxide, part number PS073 from United ChemicalTechnologies. The same effect may be achieved through the use ofpatterned hydrophilic printing inks, BSI Corporation Photolink™hydrophilic surface treatment or using CYREX injection molded part. Thinfilm materials, used for the front and back layers of the strip, arelaminated to either side of the gasket-capillary structure. The wettingagent can be applied to the channel by either an air brush or nylonbrush applicator and then dried under a heat lamp. Both methods workequally well.

Separating agents are impregnated into the matrix before, during orafter the impregnation of test reagents. The specific compounds areselected to enhance the ability of the matrix to separate whole bloodinto red blood cells and clear fluid. As discussed previously, thepreferred matrix materials comprise a microporous polyethersulfone fromGelman, Pall Hemadyne or Ahlstrom cellulose and glass media.

The separating agents which can be impregnated into the matrix may beselected from the following: polyvinyl sulfonic acid (PVSA),polyethylene glycol (PEG), polystyrene sulfonic acid (PSSA),hydroxypropyl cellulose (commercially available as Klucel™), polyvinylalcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), watersoluble salts, citrates, formates and sulfates, amino acids, chitosan(amino sugar), citric acid, phytic acid and malic acid. These materialsmay be enhanced through combining with silica or clay. The chemicalcomponents can include equivalent materials which help to separate wholeblood into red blood cells and relatively clear fluid.

Many analytes in blood exist within a narrow range. The largest normalrange for any component of whole blood is the fraction of red bloodcells in the whole blood, or hematocrit. A healthy individual may havehematocrit ratio between 35 and 55. Persons at high altitudes andnewborns often have elevated hematocrit levels, e.g., 60 or above. Sickindividuals may experience hematocrit levels of 30 or below. Anindividual with a hematocrit of 60 has a water soluble analyte, such asglucose, distributed in only 40% by volume of the whole blood sampleapplied. A 30 hematocrit blood sample is 70% liquid fraction. Thoseskilled in the art recognize the huge effect this variable compositioncan have on whole blood test results. (Many hospitals and clinical labsrely on serum analyte levels to eliminate this interference.) The bloodseparation devices and methods described in the present invention allowsfor the virtual creation of a serum system by removing the red bloodcells from the reaction area. As long as adequate fluid is delivered tothe active areas of the device, which the geometries of the deviceassure, additional clear fluid and red cells held away from the activeareas of the device do not impact the reaction. The hematocrit effect,which is a marked influence on the overall performance of most devices,is substantially eliminated in the practice of the present invention.

The indicating reagent mix must be capable of detecting the presence ofthe analyte. In general, the analyte reacts with a specific oxidaseenzyme and produces hydrogen peroxide. This strongly oxidative substancereacts with the indicator(s) present to produce a colored end product.The oxidase enzyme may be one of the following: glucose oxidase,cholesterol oxidase, uricase, alcohol oxidase, aldehyde oxidase orglycerophosphate oxidase. While the examples and preferred embodimentsherein comprise glucose oxidase in the formulations, formulation changesrequired to utilize other oxidase enzymes are evident to one who isskilled in the art. The indicator chemistries which provide acceptablecolor generation when coated on the microporous membrane(polyethersulfone) from Gelman, Pall Hemadyne or Ahlstrom Filtrationglass fiber matrix may be chosen from 3-methyl-2-benzothiazolinonehydrazone hydrachloride (MBTH) combined with 3,3-dimethylaminobenzoicacid (DMAB), MBTH combined with 3,5-dichloro-2-hydroxybenzene-sulfonicacid (DCHBS); 4-aminoantipyrene (4-AAP) (at 4 mg/ml) and5-Oxo-1-(p-sulfophenyl)-2-pyrazoline-3-carboxylic acid (OPSP); 4-AAP (at4 mg/ml) and n-(m-tolyl)-diethanolamine (NDA); 2,2'-azino-di(3-ethylbenzthiazoline) sulfonic acid (ABTS); 4 AAP (at 4 mg/ml) and4-methoxynaphthol; pyrogallol red(PGR); bromopyrogallol red (BPR); acidgreen 25 (AG); MBTH and 8-anilino-1-naphthalenesulfonate (ANS); orN-(3-sulfopropyl)aniline and MBTH; or other known and conventional dyesystem for different analytes. U.S. Pat. No. 5,306,623 to Kiser et. al.discloses effective concentrations of a number of useful dye systems.

A preferred dye system is disclosed in copending application Ser. No.08/628,794, now U.S. Pat. No. 5,776,719 filed on even date herewith andincorporated herein by reference. This preferred dye system is based onthe sulfonated form of MBTH, 3-Methyl-6-(Msulfonate)-benzothiazolinone-(2)-hydrazone (MBTH-S) where M is sodium,potassium, ammonium or other equivalent ion, but is preferably sodium.MBTH-S formed as a dye couple with DMAB, ANS or N-(3-sulfopropyl)anilineprovides an indicator system which provides a stable color end point ina short period of time. This dye system enables visual reading on areliable basis without the use of meters or complex timing sequences.

Certain indicators such as MBTH-DMAB continue to change color over time,i.e., the reaction does not reach a stable end point within a reasonabletime period. When it is desirable that such an indicator dye system isused, it is important to take the desired readings at specific timeafter wetting the test strip and beginning the reaction. U.S. Pat. No.5,049,487 to Phillips et al., incorporated herein by reference,describes the use of a change in reflectance of the matrix as a signalthat the matrix has been wetted by the sample. In the present invention,the meter design can incorporate two contacts which make contact withthe reagent impregnated test pad. When the test pad has been wetted bythe application of blood or test sample, a circuit is made and thetiming is initiated. The meter can then take readings at the appropriatetimes as required by the algorithm in the meter. Alternately, sensors inthe meter can detect an object, such as a finger or a pipette, over thetest matrix in the area to which sample is applied. The timing can beinitiated at the time of or shortly after object detection. Either ofthese approaches enables the design of a simplified, lower cost meterfor use where the indication of the dye system must be measured on atime-dependent basis.

The MBTH-ANS system described by Yu in U.S. Pat. No. 5,453,360 may beused in the methods and devices of this invention. However, bothcomponents require an acid pH of approximately 4.0, which enhancesenzyme activity and requires the use of higher levels of oxidase orperoxidase enzymes than desired in the chemistry system. A near neutralpH system is more preferred. MBTH-S dye system referred to above canexist at approximately a pH of 6 and has the advantages of being easierto formulate and enhanced enzyme activity. By using MBTH-S and ANS a dyecouple can be used which exists at a pH of 6 which permits the dyecouple dry chemistry system to be used at this higher pH. It has beenfound that the MBTH-S and N-(3-sulfopropyl)aniline formulation isanother preferred embodiment for the indicating dye system in thedevices and methods of this invention. It creates a stable end pointchemistry which is water soluble and does not sublime over time whenapplied and dried in the membrane matrix. The MBTH-S coupled with ANSprovides flat spectral absorption in the region of about 580 to 650 nm.MBTH-S coupled with ANS provides good spectral absorption, is watersoluble and does not sublime under dry chemistry storage conditions. Apreferred dye system of the MBTH-S and ANS dye couple can be used in thedevice of present invention because of the separation of the red bloodcells from the reaction site provided by the devices and methods ofpresent invention. Effective blood separation occurs using themicroporous Gelman membrane or lateral wicking Ahlstrom or Pallmaterials, making spectral absorption in the range of 580-650 nm (seeFIG. 27) acceptable. This range produces colors which are purple toblue. The lower end of the wavelength range would not be acceptable fora meter read strip if whole blood color was present in the device testarea. The use of tension modifiers, hematocrit adjustment compounds,buffers and chelators which are useful in these systems are known in theart. One who is skilled in the art can formulate an acceptable chemistrybased on the components disclosed herein and in the prior art.

The above reagents will create a chemistry which can be read with eitherby a meter or by visual color comparison. To create a visual strip whichcan be read in binary fashion as described in U.S. Pat. No. 3,964,871,issued to Hochstrasser, a plurality of test areas must be designed intothe test device. To permit the chemistry to be sensitive to thresholdlevels of analyte an antioxidant is used to inhibit or intercept thereaction in visual test zones which only change color if the analyte ispresent in greater quantity than the inhibition chemistry in that zone.They participate in a noncompetitive reaction and are consumed first bythe hydrogen peroxide. If the antioxidant is fully consumed by thereaction the dye indicator(s) is oxidized and color is developed in thetest matrix. Hochstrasser, U.S. Pat. No. 3,964,891, provides thebackground to the design and implementation of a urine inhibition teststrip. Kiser et al, U.S. Pat. No. 5,306,623, expands this for bloodtesting. Antioxidants which may be utilized include2,3,4-trihydroxybenzoic acid, propyl gallate, ascorbic acid, isoascorbicacid, 3,4 dihydroxy cinnamic acid, 3,4 dihydroxy benzaldehyde, Gallicacid and 5,6-diaminouracil. The antioxidant which is preferred in thisembodiment is ascorbic acid.

The multi-zone test systems can use various indicating reagenttechnologies:

indicating dyes and an antioxidant system to provide threshold readings,which can be utilized in multizone nonmetered test formats as describedabove,

indicating dyes which are consumed by the reaction, i.e. a test zonewith more dye will turn off at higher concentrations of analyte than atest zone with less dye, and

indicating dyes which are generated in proportion to the concentrationof an analyte, which may be used in a color match system or inconjunction with a meter.

A three level sample device can be used in the present invention basedon the chemistry systems described below.

    ______________________________________                                     Indicating Dye +           Indicating dye            increase           same amount each                        Indicating Dye +                                     concentration for    Test Zone           test area    Antioxidant  each test area    ______________________________________    Low    color match  Dye + minimal                                     Dye                        antioxidant    Medium color match  Dye + more   Dye + additional                        antioxidant than the                                     dye                        low test zone    High   color match  Dye + more   Dye + additional                        antioxidant than the                                     dye + additional                        medium test zone                                     dye    ______________________________________

The separation reagents, indicator reagents, oxidase enzymes, peroxidaseenzymes, hematocrit adjuster, buffers, antioxidants and chelatorstogether with the dye system are impregnated in a membrane matrixselected from polyethersulfone, polysulfone, polyamide, cellulose andglass fiber or Pall Hemadyne.

The issue of hematocrit level affecting the accuracy of test results isa substantial one for a test device which does not have good bloodseparation and microtitration. The membranes used in this invention canbe used in meter-read devices and, without the microtitration format,may have a larger hematocrit effect than desired. The followingembodiment of this invention can be used to compensate for thehematocrit variation of whole blood. The instrument can be designed withadditional light sources and receivers (sensors) connected to analogsignaling/conditioning circuit. These additional sensors can beimplemented so that they inspect a channel in the test device, onesensor at the beginning of the channel and one at the end. Whole bloodis applied remote from the reaction zone. The test device has acapillary channel which is clear and the movement of whole blood istimed between sensors. The time that the blood takes to travel up thecapillary is an indication of the hematocrit of the blood, and thatinformation is used to correct any shift in reflectance readings of theinstrument caused by the hematocrit level. The capillary can have twoconfigurations: a clear channel with a hydrophilic wetting agent appliedor a channel formed in a lateral wicking porous material. The layers ofpreferred embodiments of the invention are fastened with adhesive suchas 3 M grade 415 pressure sensitive acrylic adhesive. The porous inertmaterial has a low free radical content and is widely used in medicaldevices.

The various aspects of the invention disclosed herein can best beillustrated by reference to the drawings and the description thereofwhich follows.

FIGS. 1 and 2 illustrate a device of this invention which utilizes aporous matrix member 1 to achieve separation of whole blood into redblood cells and relatively clear fluid. Matrix 1 has a skin side 5 and atest side 7 and is attached to holder 49 which contains opening 21. Thematrix is preferably an intrinsically hydrophilic material and isoptionally impregnated or coated with separating reagents to facilitateand maximize blood separation. A sample of whole blood is applied to theskin side 5 of matrix 1 through opening 21. The combination of the skincharacteristics of the matrix, the hydrophilic nature and the separatingagents provide blocking of the red cells on the surface of the skin sideS while clear fluid containing the analyte flows through the skin intomatrix 1 and to test side 7. Indicator reagents are present in thematrix, as well as enzymes, hematocrit adjusters, buffers, antioxidantsand chelators, which are useful in providing a test device which iscapable of determining the level of an analyte in whole blood. Thevarious indicator reagents are known in the art conventionallyformulated into reagent cocktails in solvents and applied to matrix 1.The cocktails for each analyte to be detected are formulated into groupswhich can coexist in the same pH and solvent solutions conditions. Eachindicator or other reagent cocktail is applied to the test side 7 ofmatrix 1 and dried. When the blood sample wets the reagent present inthe matrix, the indicator in the test side of the matrix changes colorto provide the desired indication of the analyte, e.g., glucose, in theblood.

As shown in FIG. 3A, a drop of blood or other fluid 30 from the usersfinger or from an applicator may be applied to the device of FIG. 2through opening 21 and the color change may be read on test side 7 by atest instrument 72 or by visually color match. FIG. 3A illustrates atypical combination of the device of FIG. 2 as used in conjunction witha test instrument 72. When the test device of FIG. 2 is inserted in thetest instrument 72, related and necessary information may becommunicated to the test instrument 72 via an instrument readablereference code 61, shown in FIGS. 1 and 2 or by a mechanical notchpattern or a magnetic pattern shown in FIG. 3B at 101. The informationcontained in the code or pattern provides calibration data, timingsequence or other information to assure accuracy of the reading of thetest strip by the instrument. A circuit in test instrument 72 can becompleted when the blood or clear fluid from the application of drop 30wets matrix 1, connecting the contacts 70 and 71 with each other andinitiating the test sequence of the instrument or as directed byreference code 61 or pattern 101.

FIGS. 4 and 5 show a device essentially the same as FIGS. 1 and 2 buthaving multiple reaction zones or areas in matrix 1, multiple bloodapplication apertures 21 in holder 449 and multiple viewing apertures 11in support member 19. The matrix 1 has a skin side 5 and test side 7 andis impregnated with appropriate indicator reagents. The matrix 1 can beattached with adhesive to holder 449. Holder 449, matrix 1 and support19 are laminated to form the device shown in FIG. 5. Blood samples areapplied to skin side 5 of matrix 1 through apertures 21 in the holder449 and the color change is observed on the test side 7 of matrix 1through openings 11 in support member 19. FIG. 39 shows a similar devicebut with individual, discrete matrix members for each test zone or setof opening. In this embodiment the skin side 5 can include the top andsides of each discrete matrix element to prevent red blood cells fromentering a matrix element.

It is to be understood that in all embodiments of this invention where amember is called for as having openings therein for visual reading ormeter measurement of the indication of the indicator, it is intendedthat these are visual or transparent openings. Thus, such a member maybe a solid sheet with no physical openings or holes therein, butprovides visual or meter access to the indicator by being transparent orsufficiently translucent at least at the appropriate test sites forreading the indicator indications, or may be entirely transparent. Sucha member can also be a composite laminate of an opaque layer, such asaluminum foil, with opening therein and a transparent plastic film, withopening therein and a transparent plastic film that is a solid sheet butprovides visual access through the openings in the opaque layer.

FIGS. 6 and 7 show a device essentially the same as FIGS. 4 and 5 buthaving a blood delivery system for distributing a blood sampleinternally in the device. The blood delivery system is comprised ofgasket layer 13 containing openings 621, laminated to a channel layer 23containing capillary passageway 25 communicating with notches 33 whichform reservoirs above openings 621. Blood is applied to the devicethrough sample receiving opening 29 in cover member 31. The bloodtravels through the capillary passageway 25 which flow may be assistedby a wetting agent applied to the bottom thereof. The capillarypassageway 25 is vented by cut outs 24 in the channel layer 23, whichcommunicates with vent 22 in gasket layer 13. Blood fills the notches 33forming reservoirs in the channel layer 23 and passes through openings621 to the skin side 5 of matrix 1. Channel layer 23 and gasket layer 13can be coated with a wetting agent to aid in blood flow through thechannel or can be an inherently hydrophilic plastic, such as asulfonated plastic. The blood is separated into relatively clear fluidwhich is passed through skin side 5 to test side 7 of matrix 1 and redblood cells which are retained on the surface of skin side 5. The colorformed in the indicator in the test side 7 of matrix 1 is viewed throughopenings 11 in support member 19. The device of FIGS. 6 and 7 is made bylaminating cover member 31, channel layer 23, gasket layer 13, matrix 1and support 19 to form a unitary device. Appropriate adhesives may beused between the various layers to provide adhesion of the layers intothe formation of the unitary device of FIG. 7 and to provide appropriatesealing of the multiple test zones from each other and to provide aconfined internal path for the blood sample to flow from opening 29through the confined path defined the capillary passageway 25, notches33, openings 621 to the skin side 5 of matrix 1 and prevent any flow offluid from one test zone to another. In this manner each individual testarea defined by openings 11 and support member 19 can be observedaccording to the indicator present in the corresponding zone of the testside 7 of matrix 1. It may be desirable to have different indicatorreagents present in each different test zone. Alternatively it may bedesirable to have a graduated concentration of indicator reagentdifferent indicator reagents along the length of matrix 1 whereby thecolor change in openings 11 will be graduated for a given blood sampleto provide the desired reading or measurement from the indicator orindicators present.

FIGS. 8A, 8 B and 9 illustrate the devices constituting another aspectof the present invention. In this aspect a substantially noncompressiblemember 93 is provided with opening 91 having a predetermined volumetricsize. A matrix member 1 having a skin side 5 and test side 7 as shown inFIG. 1 is compressed against member 93 such that a portion of matrix 1protrudes or extends into opening 91 and the remaining portion of matrix1 is compressed to a thinner layer as illustrated in FIG. 8B. Forillustration purposes FIG. 8B shows partially compressed matrix member 1separated from member 93. However, it is understood once matrix member 1is compressed against member 93 and into opening 91 it need not beseparated from member 93 but may be positioned directly on holder 9 toresult in the unitary device illustrated in FIG. 9. In this device theskin side of matrix 1 which protrudes into opening 91 of member 93remains exposed for application of a blood sample in opening 91 with thetest side of matrix 1 being visible for reading or measurement throughopening 96 and holder 9. It is further to be understood that theorientation of matrix 1 may be reversed in this device whereby the testside 7 of matrix 1 is compressed against member 93 and protrudes intoopening 91 leaving the skin side of matrix 1 to contact holder 9. Insuch reverse configuration the blood sample can be applied throughopening 96 to the skin side 5 of matrix 1 then the reading ormeasurement of sample side 7 of matrix 1 can be performed throughopening 91. As illustrated in FIGS. 1, 2 and 3, the device of FIGS. 8A,8 B and 9 can contain machine readable coding 62 for calibration orcontrol of a test instrument as indicated above.

Another embodiment of this aspect of the invention is illustrated inFIG. 8A, after matrix member 1 is compressed against member 93 to formthe protrusion of matrix member 1 into opening 91 the partiallycompressed matrix 1 can be removed from member 93 and placed on holder 9as shown in FIG. 8A. In this embodiment the protruding noncompressedportion of partially compressed matrix 1 is inserted into opening 96 toprovide a simple device on which a blood sample may be applied toopening 96 and skin side 5 of matrix 1 and the indicator read ormeasured on test side 7 of matrix 1. It will further be apparent andunderstood that in making the device of FIG. 9 matrix 1 can becompressed between member 93 and holder 9 in an appropriate laminationprocess with appropriate adhesives. In such a process opening 96 istemporarily blocked with a tool to prevent matrix 1 from protruding intoopening 96 during the lamination and compression.

An important aspect of the device shown in FIG. 8B and 9 is that opening91 is provided to have a predetermined volumetric size. This volumetricopening is substantially filled with the protruding portion of matrix 1containing an indicator reagent. This configuration thereby provides aspecific known and predetermined volume in opening 91 which provides amicrotitration chamber of a given volume for a given quantity ofindicator in the protruding matrix 1 positioned within volumetricopening 91. Thus, in addition to an ordinary color indication by anindicator, this device can provide a specific, concentration indicationon a titration basis for a known volume of fluid filling volumetricopening 91 and a given amount or concentration of indicator or otherreagent present in volumetric opening 91.

As described above a blood sample applied to the device of FIG. 9 or thedevice of FIG. 8A is applied to the skin side of matrix 1 present involumetric opening 91 or volumetric opening 96, whereby the red bloodcells or other solids are blocked from passage by skin side 5 and theblood fluids are passed through skin side 5 to test side 7 of matrix 1.

FIGS. 10A and 10B show a device essentially the same as FIGS. 8B and 9but having multiple volumetric openings 91 with multiple portions ofmatrix 1 protruding therein. The construction and use of the devices ofFIGS. 10A and 11 are the same as for the devices of FIGS. 8B and 9except that multiple test zones are provided. FIG. 11 is a illustrationof an alternative configuration showing that the portions of matrix 1which protrude into volumetric openings 91 may be rounded in nature asopposed to exactly conforming to the full volume of volumetric opening91. It is not necessary for the portion of matrix 1 protruding intovolumetric opening 91 to completely fill the available volume ofvolumetric opening 91. It is simply necessary that the amount or portionof matrix 1 which does protrude into volumetric opening 91 whencompressed against member 93 be known and calibrated in order thataccurate volumetric titration tests can be performed for the givenvolume of volumetric opening 91 and a given amount of indicator orreagent present in the matrix protruding into volumetric opening 91. Inuse the volumetric opening 91 can be filled with a given and knownvolume of test fluid whether or not the volumetric opening 91 iscompletely filled with the protruding portion of matrix 1.

The device of FIGS. 12 and 13 are essentially the same as the devices ofFIGS. 10A and 10B but with the added feature of an internal capillarypassageway for distributing a blood sample internally to the varioustest zones, as described above in connection with the device shown inFIGS. 6 and 7. In use the sample enters opening 29, flows throughcapillary passageway 25 to notches 33 and openings 621 to the skin side5 of matrix 1. The fluid portion passes through skin side 5 to test side7 and the indicator, which is read or measured through openings 11.

FIGS. 14, 15 and 16 illustrate a modification of the device of FIGS. 12and 13 wherein the layout of the test sites are configured such that theflow of blood through capillary 25 can be aided by gravity flow. In thisdevice the blood sample is introduced through opening 29 in cover member31. The blood can flow through capillary passageway 25 and openings 33to contact the skin side 5 of the protrusions of matrix 1 positioned inopening in member 35. When the assembled device of FIG. 14 is placed onits edge with opening 29 at the top edge it can be seen that gravitywill assist in the flow of the blood along capillary passageway 25 andthrough notches 33 and vents 36. FIG. 16 shows support member 19 havinga corresponding arrangement of openings 11 to correspond to the layoutof the protrusions of the matrix member 1. FIGS. 37 and 38 show the samedevice with user instructions on one side, i.e., where to apply theblood sample, and indicia on the other side for visual indication of thetest results, i.e., level of glucose concentration.

FIGS. 17, 18, 19 and 20 illustrate a variation of the device of FIGS. 12and 13. In this configuration matrix 1 is compressed against member 34as shown in FIG. 17, which results, after removal of the compressedmatrix 1 from member 35 in a partially compressed matrix 1 having aprotrusion of uncompressed portion of matrix 1 as shown in FIG. 18. FIG.19 illustrates the remaining uncompressed portion of matrix 1 after mostof the compressed portion of matrix 1 has been removed from around theuncompressed portion leaving element 17 which is a uncompressed shape ofmatrix 1 having a small border around the base thereof of compressedmatrix 1. These elements may then be assembled into appropriate openingssuch as the volumetric openings 91 illustrated in FIG. 10A. As shown inFIG. 20 the matrix elements 17 may be assembled so that they fit intoopenings in member 35 and sealed by adhesive around the border at thebase of each matrix element. This type of device can be assembled andused as described above with respect to the devices of FIGS. 12, 13 and14.

FIGS. 21 and 22 illustrate another aspect of the present inventionwherein the porous matrix is utilized in a device having an offsetconfiguration. This device provides for the lateral transfer of thefluid sample through the matrix member to provide certain advantages inthe reading or measurement of the indicators. As shown in FIG. 21 holder49 contains opening 21, matrix 40 is positioned between holder 49 andsupport 19, and support 19 contains opening 11 which is laterally offseta given distance from opening 21 in holder 49. In this device matrix 40has initial area 47 corresponding to opening 21 and test area 45corresponding to opening 11. A sample fluid is introduced throughopening 21 into initial area 47 of matrix 40, passes laterally throughmatrix 40 into test area 45 and reacts with the indicator, which can beread or measured through opening 11. Matrix 40 is a porous matrixcontaining pores capable of blocking in the lateral distance between theinitial area 47 of matrix 40 and test area 45 the passage of solids butis capable of allowing the passage of fluid from initial area 47 to testarea 45. In this device the matrix material 40 provides a separation ofthe solids such as red blood cells over the lateral distance frominitial area 47 to test are 45 such that the indicator present in testarea 45 as viewed through opening 11 will be substantially free fromsolids or red blood cells which may interfere with the indicationprovided by the indicator reagents in the test area 45 of matrix 40.

FIGS. 23 and 24 illustrate a device similar to FIGS. 21 and 22 whereinmatrix member 40 is compressed against member 93 whereby a portion ofmatrix member 40 is compressed against the surface of 93 and a portionof matrix member 40 protrudes into opening 91 of member 93. Thisconfiguration is similar to that described above with respect to FIG. 8Band 9, except in this configuration the opening 91 is elongated thusproviding an elongated protrusion of uncompressed portion of matrix 40in order to provide an uncompressed portion of matrix 40 extending fromopening 21 in holder 49 to the offset location of opening 11 in supportmember 19. Thus FIG. 24 shows the assembled device with member 93 andmatrix 40 positioned between holder 49 and support member 19. In thisdevice a fluid sample in introduced in opening 21 where it passesthrough initial area 47 of matrix 40 and flows laterally through matrix40 to test area 45 which is positioned to correspond with opening 11. Asdescribed above in the lateral distance between initial area 47 and testarea 45 the porous nature of matrix 40 is capable of blocking thepassage of solids, such as red blood cells, and allowing passage offluid to test area 45 to react with indicator present in test area 45,which indication can then be viewed through opening 11.

The device of FIGS. 25 and 26 is essentially the same as the deviceillustrated and described in FIGS. 21 and 22 but having multiplereaction zones. Otherwise the configuration is similar in that openings21 correspond to initial area 47 and openings 11 correspond to test area45 of matrix 40. The function of the device of FIGS. 25 and 26 is thesame as the device FIGS. 21 and 22 but on a multiple zone basis.

The device of FIG. 27 is essentially the same as the device illustratedand described in FIGS. 23 and 24 except in a multiple test zoneconfiguration. Similarly the device of FIGS. 28 and 29 correspond to thedevice of FIGS. 25 and 26 but further incorporating the internalcapillary passageway distribution system for the fluid as describedabove in connection with FIGS. 6 and 7. Similarly, FIG. 30 illustrates adevice of FIG. 27 but with internal capillary passageway distributionsystem for the fluid.

FIGS. 31 and 32 illustrate another aspect of the devices of the presentinvention which enable analysis of an analyte in a fluid by measuringthe initial flow rate through a restricted flow passageway. In thisdevice member 323 contains first opening 322 and second opening 366 withrestricted flow passageway 325 communicating with the first opening andthe second opening, whereby the fluid sample introduced into the firstopening 322 will flow by capillary action through passageway 325 toopening 366. The device further comprises cover layer 331 having opening321 corresponding to opening 322. The device further comprisestransparent support member 319 having opening 311 corresponding toopening 366, which can optionally have matrix member 1 compressed intoor preshaped to fit into opening 366. In this device support member 319is transparent so that the flow of fluid from opening 322 throughpassageway 325 to opening 366 can be observed and can be measured bydetector 64. Detector 64 is adapted to measure the rate at which theinitial flow of fluid occurs from opening 322 through passageway 325.The rate of flow of the fluid can be correlated to known concentrationsof an analyte in the fluid so that measuring the rate of initial flow ofa know fluid for a known analyte will provide the concentration of theanalyte in the fluid being tested. When the fluid reaches opening 366and flows into matrix 1 containing appropriate indicator reagents, thetypical reaction will occur and the indication of the indicator can beobserved or measured through opening 311 and support member 319. Thisconfiguration presents several distinct advantages. The sample may beapplied to the test strip away from the reading area, which may limitbiohazard exposure if the meter is used with multiple patients. The rateof travel through the capillary corresponds to the hematocrit of theblood sample. By calculating the lag as the blood moves from one pointin the channel to another, a hematocrit may be determined. Ifappropriate, a hematocrit correction factor may be applied to the testresult, improving overall system performance.

FIG. 33 is an illustration of a device which is a variation of thedevice illustrated and described in FIGS. 31 and 32. In this devicepassageway 325 contains a portion of matrix 1 which is portion 67corresponding to the shape of passageway 325. In this configuration ofthe device the flow rate of fluid from opening 322 to opening 366 willbe observed and measured through transparent member 319 as it flowsthrough matrix 67 to opening 366. As with the device in FIGS. 31 and 32the initial flow rate of a particular fluid through passageway 325containing matrix 67 can be correlated to the flow of fluid through theidentical device for known concentration of the analyte of interest.

FIGS. 34, 35 and 36 illustrate a device similar to the devices shown anddescribed in FIGS. 10A and 20, wherein. The passageway for internaldistribution of the fluid is contained on the bottom side of covermember 31 wherein channel 340 communicates with opening 29 and withopenings 91 and member 93. In this device fluid sample enters opening 29in cover member 31 and flows laterally through channel 340 (shown inbottom view of cover member 31 in FIG. 36) to each of the opening 91 andmember 93 where the fluid contacts each of matrix elements 37. The fluidflows through skin side 5 and into matrix elements 37 containing theindicator reagent. Thus the indication of the indicator can be viewedmeasured through openings 11 in support member 19.

In general the matrix material 1 such as illustrated in FIGS. 1 and 2will generally by in the ranges of about 3 mils to 7 mils in thickness.(1 mil=0.001 inch=0.0254 mm.) In most test devices a thickness of about4 to 5 mils is preferred. On the skin side 5 of matrix 1, the thicknessof the skin capable of blocking the passage of red blood cells will beabout 0.5 mil or less. The holder member such as 49 in FIGS. 1 and 2will generally be a polymeric strip having a thickness from about 5 milsto about 12 mils in most applications and depending on the type ofpolymeric strip employed a thickness of about 7 to 8 mils is preferredfor the holder member. The support member such as 19 in FIG. 4 can alsohave a thickness of from about 5 mils to about 12 mils with about 7 to 8mils in thickness being preferred when the support member is polymeric.The support member may also be made of a metal foil such as aluminumfoil in which case the support member may have a thickness of about 1 to3 mils in thickness. It will be apparent that when the support member isa metal foil it may be laminated with a transparent plastic film wherethe openings in the metal foil are appropriately positioned and thetransparent film is laminated between the foil and the matrix memberwhere the transparent polymeric film can provide protection of thematrix member containing the indicator reagent from contamination. Itwill further be recognized that a support member can also be atransparent polymeric strip where the openings are merely visuallytransparent areas which allow reading or measurement of the indicationof the indicator on the matrix member.

Certain member of the devices of this invention such as 93, 13 and 35which provide fixed volumetric openings into which the matrix materialis compressed will generally be in the range of 4 to 12 mils inthickness and preferably about 4 to 5 mils in thickness. It will also berecognized that these members providing the volumetric fixed sizeopenings will preferably be injection molder materials but can besufficiently rigid in noncompressible polymeric strips from which thedesired volumetric opening has been punched or dye cut.

It will be recognized by those skilled in the art that the overallthickness of the assembled test strip devices according to thisinvention may vary according to the desired use. The overall thicknessof the assembled devices can range from about 8 to about 40 mils. Due tothe strength provided by laminating the various layers thinner layeredmaterials may be used and provide sufficient strength. However, theoverall thickness of a test strip device according to this inventionwill be determined also by the necessary and desired thickness of thematrix member to provide color separation and sufficient volumeabsorption. In addition the embodiments of this invention providing thefixed volumetric openings will dictate the thickness of the layersproviding the volumetric openings of desired volume for the titrationtests enabled by the devices of this invention.

When the matrix member is compressed into the adjacent member as inFIGS. 8A, 8 B and 9, the typical matrix material having a thickness ofabout 5 to about 12 mils will be compressed in the compressed area to athickness of about 1 mil or less and typically less than about 0.5 milAt the same time the portion of the matrix layer which protrudes intothe volumetric opening will remain at or near its full originalthickness.

In the embodiments of the devices according to FIGS. 31 and 33, therestricted flow capillary passageway will typically be about 5 to 25mils in length. The length of the passageway will be determined by theoptical detectors used to detect and measure the initial flow rate offluid through the passageway and will be determined by the nature of theflow rate and flow pattern of the fluid being detected in thepassageway. Typically a passageway of 5 to 10 mils in length issufficient to measure the initial flow rate of the fluid through thepassageway. It has been found that for a plastic channel treated with adimethyl siloxane ethylene oxide wetting agent the cross section shouldbe about 5 mils by about 40 mils but can be as small as about 5 mils byabout 25 mils. Similar size channel is useful in a plastic memberwithout a wetting agent provided the plastic inherently hydrophilic.

The methods of assembling the devices according to the present inventionwill be apparent to one skilled in the art following the teachingcontained herein together with conventional laminating techniques forapplication of adhesive to the various layers, heat bonding variouslayers and similar techniques for assembly of the devices disclosedherein.

The devices of this invention are conveniently made into test strips ofconvenient size and configuration particularly for individual use byvisual inspection or for use in instruments or meters which are adaptedto measure the color or other indication provided by the test strips. Itis also convenient to provide the test strip devices of the presentinvention in a kit form for use by an individual wherein the kitcontains a test strip according to the present invention, an antisepticapplicator, an anesthetic applicator, a sharp article for puncturing theskin or the individual to provide a blood sample, and a bandage for theskin puncture site. When supplied in this kit form, proper andconsistent use by the individual will be encouraged and facilitated dueto the convenience of the kit.

It is desirable to have a system, or kit, which contains all thenecessary supplies for performing a test. This is particularlyadvantageous for diabetics, many of whom are highly mobile. Thisinvention describes a visual test strip which lends itself well toincorporation in a kit. An individually foil wrapped strip coupled witha commercially available disposable lancing device provides the minimumsupplies required to perform a blood glucose test. The kit mayoptionally include a prepackaged towlette to clean and/or numb the testarea and an adhesive bandage to cover the lanced site. The presentationof a complete testing kit is extremely useful for individuals as well asfor clinics or visiting nurse groups where complete segregation of alltesting supplies from patient to patient is advantageous.

One example of a material useful for lateral transfer of fluidcontaining analyte and blocking lateral transfer of solids is acomposite cellulose and glass fiber matrix, such as that available fromAhlstrom as part number 1661 or 1662, especially to separate the wholeblood into red blood cells and substantially clear fluid. Anotherexample is Pall Hemadyne membrane. The whole blood is applied to thematrix and wicks laterally into the matrix material. As the samplewicks, the red blood cells adhere to the glass fibers or other matrixfibers and the clear fluid moves laterally into the test area where thedry reagents are present. The reagents in the test area of the matrixare rehydrated by the clear fluid component of the whole blood and arethen able to indicate the presence and concentration of one or moreanalytes of interest. Separating agents impregnated into the matrix canassist with the separation of red blood cells and facilitate the wickingof the substantially clear fluid into the test area. This configurationcoupled with microtitration devices and methods described above willproduce an accurate test device.

The following is an example of making and using the devices of thisinvention.

EXAMPLES

Glucose Test

    ______________________________________    Example A: Test Reagents    ______________________________________    Reagent 1a            40 mg MBTH-S            80 mg DMAB            5 ml acetonitrile and 5 ml water            Stir until all solids are dissolved.    Reagent 2a            6 ml water            10 mg EDTA, disodium salt            200 mg PolyPep, low viscosity (Sigma)            0.668 g sodium citrate            0.523 g citric acid as a hematocrit adjuster            0.2 M Aconitic acid buffer            3% polyethylene glycol (PEG), as a separating agent            0.5% Polyquart, a binder            2.0 ml 6 wt % Gantrez AN-139 dissolved in water (GAF)            30 mg horseradish peroxidase, 100 units/mg, and 3.0            glucose oxidase, 200 units/ml            Stir until dissolved.    Reagent 3a            Antioxidant solution of 50:50 ethanol and ascorbic acid at            a pH of 4.0, in varying amounts.    ______________________________________    Example B: Test Reagents    ______________________________________    Reagent 1b            20 ml water            420 mg citric acid (a buffering agent). Adjust th pH of            the citric acid solution with NaOH to a value of 4.25.            16.7 mg EDTA            90 mg Gantrez S95 available from GAF            250 mg Crotein SPA            20,500 units glucose oxidase            16,200 units peroxidase    Reagent 2b            10 ml of a mixture of 3 parts by volume water and 7 parts            by volume isopropyl alcohol            13 mg MBTH-S            40 mg ANS    Reagent 3b            Antioxidant solution of ethanol and ascorbic acid in            varying amounts.    Test A    Polyethersulfone matrix    ______________________________________

A piece of polyethersulfone membrane is uniformly coated with reagent1a; the excess is squeegied off and the material is dried. The membraneis then coated with reagent 2a in the same fashion and dried. Theantioxidant solution reagent 3a is directly applied to the test areas invarying concentrations using a syringe. The membrane is then assembledinto a test device as shown in FIG. 2. Whole blood is applied to thesample opening and the glucose level is read from the front based on theindicator response in each of the test zones.

Cellulose and Glass Fiber

A piece of cellulose and glass fiber matrix is discretely coated withreagent 1a and dried. It is then discretely coated with reagent 2a anddried. The antioxidant solution reagent 3a is applied to each test areain varying concentrations using a syringe. The membrane is thenassembled into a test device as shown in FIG. 21. Whole blood is appliedto the sample opening and the glucose level is read from the opening onthe opposite side.

Pall Hemadyne Membrane

A piece of Pall Hemadyne membrane is uniformly coated with reagent 1a,excess fluid is squeegied off and the material is dried. It is thenuniformly coated with reagent 2a in similar fashion and dried. Theantioxidant solution reagent 3a is applied discretely to each test areain varying concentrations using a syringe. Whole blood is applied to thesample opening and the glucose level is read from the front.

Test B

Polyethersulfone matrix

A piece of polyethersulfone membrane is uniformly coated with reagent1b, the excess is squeegied off and the material is dried. It is thencoated with reagent 2b in the same fashion and dried. The antioxidantsolution reagent 3b is applied to the test areas in varyingconcentrations using a syringe. The membrane is then assembled into atest device as shown in FIG. 2. Whole blood is applied to the sampleopening and the glucose level is read from the front based on theindicator response.

Cellulose and Glass Fiber

A piece of cellulose and glass fiber matrix is discretely coated withreagent 1b and dried. It is then discretely coated with reagent 2b anddried. The antioxidant solution reagent 3b is applied to each test areain varying concentrations using a syringe. The membrane is thenassembled into a test device as shown in FIG. 21. Whole blood is appliedto the sample opening and the glucose level is read from the front.

Pall Hemadyne Membrane

A piece of Pall Hemadyne membrane is uniformly coated with reagent 1b,excess fluid is squeegied off and the material is dried. It is thenuniformly coated with reagent 2b in similar fashion and dried. Theantioxidant solution reagent 3b is applied discretely to each test areain varying concentrations using a syringe. Whole blood is applied to thesample hole and the glucose level is read from the front.

The dry chemistry reagent system can be used with the identifiedmembranes in many different ways. The system can be used to develop avisual strip for multiple analytes or for varying concentrations of thesame analyte. The system can be used for meter read or color matchtests. Additional enhancements can be developed by interfacing thestrips with a meter and providing novel interface systems for the testdevice and meter. The following systems could be incorporated into atest device to provide calibration information and start of testsignals:

Barcode on strip

Magnetic strip

Notches or magnetic printed areas in the handle of the strip whichinterface with contacts or reed switches in the meter to provide abinary value, i.e. a 1 equals present and a 0 equals not present. Thus,16 different settings can be coded into the strip as follows.

    ______________________________________    Value   Notch A  Notch B    Notch C                                       Notch D    ______________________________________    0       0        0          0      0    1       1        0          0      0    2       0        1          0      0    3       1        1          0      0    4       0        0          1      0    5       1        0          1      0    6       0        1          1      0    7       1        1          1      0    8       0        0          0      1    9       1        0          0      1    10      0        1          0      1    11      1        1          0      1    12      0        0          1      1    13      1        0          1      1    14      0        1          1      1    15      1        1          1      1    ______________________________________

We claim:
 1. A method for determining a corrected concentration of an analyte present in a fluid comprising:providing a device comprising:a member comprising a first opening for receiving a fluid sample and a second opening for receiving fluid from the first opening; a restricted passageway communicating with the first opening and the second opening whereby the fluid sample flows from the first opening to the second opening; a detector for measuring a rate of flow of the fluid sample through the restricted passageway; and a porous matrix positioned in the second opening which comprises an indicator regent for measuring the concentration of the analyte in the fluid sample; applying the fluid to the first opening; measuring the rate of flow of the fluid through the passageway; measuring the analyte concentration from the indicator reagent; correlating said rate of flow to known hematocrit values; calculating a hematocrit correction factor from the correlation; and applying the hematocrit correction factor to the measured analyte concentration in the fluid to obtain a corrected concentration of the analyte.
 2. A method according to claim 1 wherein the porous matrix comprises a polyethersulfone membrane.
 3. A method according to claim 1 wherein the porous matrix comprises a cellulose and fiber glass matrix.
 4. A method according to claim 1 Wherein the porous matrix and indicator regent is positioned in said second opening having a predetermined volume.
 5. A method according to claim 2 wherein the porous matrix and indicator reagent is positioned in said second opening having a predetermined volume.
 6. A method according to claim 3 wherein the porous matrix and indicator reagent is positioned in said second opening having a predetermined volume. 